Fluorescence technology is enjoying ever-increasing interest from chemistry to many areas of biology. In certain instances, fluorescent molecules are used to detect the presence of analytes in food and environmental samples. Some sensitive and quantitative fluorescence detection devices are ideal for in vitro biochemical assays such as DNA sequencing and blood glucose quantification. Moreover, certain fluorescent probes are indispensable for tracing molecular and physiological events in living cells. Finally, fluorescence measurements are often used in many high-throughput screenings.
The primary advantages of fluorescence technology over other types of optical measurements include sensitivity, simplicity, and a wealth of molecular information. Fluorescence measurements are highly sensitive because of the generally low level of fluorescence background in most chemical and biological samples. Along with the advances in fluorescence instrumentation such as confocal and multi-photo fluorescence microscopies, three-dimensional imagings of cellular events and biological species dynamics have become possible in real-time.
Particularly, fluorescence in biological sciences is generally used as a non-destructive way for tracking or analyzing biological molecules, such as proteins, metal ions, reactive oxygen species (ROS)/reactive nitrogen species (RNS), and so on, by recording or imaging the fluorescence emission of certain fluorescent probes for corresponding biological molecules at specific wavelengths where there is no cellular intrinsic fluorescence induced by the excitation light.
Among these intriguing biological molecules in living systems, reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been receiving much attention from the scientific community in the development of fluorescent probes for their detection in biological samples. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generally known to scientists as very small inorganic or organic molecules with high reactivity in living systems. There are various forms of ROS and RNS, including free radicals such as superoxide radical, hydroxyl radical, nitric oxide, nitrogen dioxide, and organic peroxyl radical; as well as non-radical species such as hydrogen peroxide, singlet oxygen, ozone, nitrous acid, peroxynitrite, and hypochlorite. ROS and RNS are by-products of cellular respiration. Under normal conditions, ROS and RNS are present in very low levels and play important roles in cell signaling; while during oxidative stresses, ROS and RNS levels increase dramatically, which can cause serious damages to various biological molecules such as protein, lipids and DNA. The excessive generation of ROS and RNS has been implicated in a lot of human diseases, such as cardiovascular diseases, inflammatory diseases, metabolic diseases, cancer and central nervous system diseases. Therefore, there is a strong need for chemicals that can sensitively and selectively measure, detect or screen certain ROS and RNS to address their physiological roles both in vitro and in vivo.
Peroxynitrite has the strongest oxidizing power among the various forms of ROS and RNS, and their selective detections are highly desirable to clearly explain their critical roles in living organisms. Peroxynitrite (ONOO−) is a short-lived oxidant species that is formed in vivo by the diffusion-controlled reaction (k=0.4-1.9×1010 M−1s−1) of nitric oxide (NO) and superoxide (O2.−) in one to one stoichiometry. The oxidant reactivity of peroxynitrite is highly pH-dependent and both peroxynitrite anion and its protonated form peroxynitrous acid can participate directly in one- and two-electron oxidation reactions with biomolecules. The pathological activity of ONOO− is also related to its reaction with the biologically ubiquitous CO2, thereby producing the highly reactive radicals CO3−. and NO2. in about 35% yield. As a result of this, peroxynitrite can nitrate tyrosine and oxidize proteins, lipids and iron and sulfur clusters of biological molecules. Like other oxidizing agents in living organisms, peroxynitrite and its protonated form have been associated with both beneficial and harmful effects. However, several studies have implicated that peroxynitrite contributes to tissue injury in a number of human diseases such as ischemic reperfusion injury, rheumatoid arthritis, septic shock, multiple sclerosis, atherosclerosis, stroke, inflammatory bowl disease, cancer, and several neurodegenerative diseases (MacMillan-Crow, L. A. et al., Proc. Natl. Acad. Sci. USA 1996, 93, 11853-11858; Rodenas, J. et al., Free Radical. Biol. & Med. 2000, 28, 374; Cuzzocrea, S. et al., Pharmacol Rev. 2001, 53, 135-159; Szabo, C. Toxicol. Lett. 2003, 140, 105-112; White, C. R. et al., Proc. Natl. Acad. Sci. USA 1994, 91, 1044-1048; Lipton, S. A. et al., Nature 1993, 364, 626-632; Pappolla, M. A. et al., J. Neural Transm. 2000, 107, 203-231; Beal, M. F., Free Radical Biol. & Med. 2002, 32, 797-803).
At present, peroxynitrite probes with green fluorescent color are available (U.S. patent application Ser. No. 12/417,672); however, the existing green fluorescent probes exhibit limited intracellular retention in cell assays. In addition, peroxynitrite probes with other fluorescent colors or with the ability to localize in the desired intracellular compartment are rare. Long-wavelength fluorogenic probes, such as yellow, red, far-red, and near-infrared (NIR) fluorogenic probes, are more attractive and advantageous than green probes for providing reliable imaging in biological samples, since they effectively avoid the interference from the auto-fluorescence of cells in the green region and possess longer excitation/emission wavelengths with deeper penetration into cells and tissues. Therefore, new generations of fluorescent probes with much more desirable and reliable detection and imaging of peroxynitrite are needed.